Surface damage in cystine, an amino acid dimer, induced by keV ions.

We have studied the interaction of an ion beam (17.6 keV F-) with cystine, a dimer formed by the binding of two cysteine residues. Cystine can be considered as an ideal prototype for the study of the relevance of the disulfide (-S-S-) chemical bond in biomolecules. For the sake of comparison, the amino acid cysteine has also been subjected to the same experimental conditions. Characterization of the samples by XPS and NEXAFS shows that both pristine cystine and pristine cysteine are found as a dipolar ion (zwitterion). Following irradiation, the dimer and the amino acid show a tendency to change from the dipole ion form to the normal uncharged form. The largest spectral modification was observed in the high resolution XPS spectra obtained at around the N 1s core level for the two biomolecules. The 2p sulfur edge spectra of cysteine and cystine were much less sensitive to radiation effects. We suggest that the disulfide bond (-S-S-) remains stable before and after irradiation, contributing to the larger radiation stability of cystine as compared to the amino acid cysteine.

VUV and soft x-ray ionization of a plant volatile: Vanillin (C8H8O3).

Plant volatiles are emitted by plants in response to several forms of stress, including interaction with energetic photons. In the present work, we discuss the interaction of extreme UV and soft X-ray photons with a plant volatile, vanillin. The single and double (multiple) ionization of the vanillin molecule have been studied for the first time using time-of-flight mass spectrometry and VUV and soft X-ray photons (synchrotron radiation, at 12.0 eV, 21.2 eV, 130 eV, 310 eV, 531 eV, and 550 eV). At 12.0 and 21.2 eV, only singly charged species are observed and the parent ion, C8H8O3 (+), is the dominant species. Energy differences for some selected fragments were calculated theoretically in this energy region. At 130 eV, direct double and triple ionization of the valence electrons may occur. The fragmentation increases and CHO(+) becomes one of the main cations in the mass spectrum. The molecular ion is still the dominant species, but other fragments, such as C6H5O(+), begin to present similar intensities. At 310 eV, C 1s electrons may be ionized and Auger processes give rise to dissociative doubly ionized cations. Ionization around the O 1s edge has been studied both at the 531 eV resonance and above the ionization edge. Resonant and normal Auger processes play a significant role in each case and a large fragmentation of the molecule is observed at both photon energies, with intense fragments such as CHO(+) and CH3 (+) being clearly observed. A near edge X-ray absorption fine structure spectrum of the vanillin molecule was obtained around the O 1s ionization threshold. In addition, the fragmentation of vanillin has also been studied using a fast beam of electrons (800 eV), for the sake of comparison.

Application of a multivariate normal distribution methodology to the dissociation of doubly ionized molecules: The DMDS (CH3 -SS-CH3 ) case.

RATIONALE: The ion-ion-coincidence mass spectroscopy technique brings useful information about the fragmentation dynamics of doubly and multiply charged ionic species. We advocate the use of a matrix-parameter methodology in order to represent and interpret the entire ion-ion spectra associated with the ionic dissociation of doubly charged molecules. This method makes it possible, among other things, to infer fragmentation processes and to extract information about overlapped ion-ion coincidences. This important piece of information is difficult to obtain from other previously described methodologies. METHODS: A Wiley-McLaren time-of-flight mass spectrometer was used to discriminate the positively charged fragment ions resulting from the sample ionization by a pulsed 800 eV electron beam. We exemplify the application of this methodology by analyzing the fragmentation and ionic dissociation of the dimethyl disulfide (DMDS) molecule as induced by fast electrons. The doubly charged dissociation was analyzed using the Multivariate Normal Distribution. RESULTS: The ion-ion spectrum of the DMDS molecule was obtained at an incident electron energy of 800 eV and was matrix represented using the Multivariate Distribution theory. The proposed methodology allows us to distinguish information among [CHn SHn ]+ /[CH3 ]+ (n = 1-3) fragment ions in the ion-ion coincidence spectra using ion-ion coincidence data. Using the momenta balance methodology for the inferred parameters, a secondary decay mechanism is proposed for the [CHS]+ ion formation. As an additional check on the methodology, previously published data on the SiF4 molecule was re-analyzed with the present methodology and the results were shown to be statistically equivalent. CONCLUSIONS: The use of a Multivariate Normal Distribution allows for the representation of the whole ion-ion mass spectrum of doubly or multiply ionized molecules as a combination of parameters and the extraction of information among overlapped data. We have successfully applied this methodology to the analysis of the fragmentation of the DMDS molecule. Copyright © 2015 John Wiley & Sons, Ltd.

Single and double ionization of the camphor molecule excited around the C 1s edge.

RATIONALE: An interesting class of volatile compounds, the monoterpenes, is present in some plants although their functions are not yet fully understood. We have studied the interaction of the camphor molecule with monochromatic high-energy photons (synchrotron radiation) using time-of-flight mass spectrometry and coincidence techniques. METHODS: A commercial sample of S-camphor was admitted into the vacuum chamber, without purification, through an inlet system. Monochromatic light with energy around the C 1s edge was generated by the TGM beamline at the Brazilian Synchrotron Facility. A Wiley-McLaren mass spectrometer was used to characterize and detect the ions formed by the camphor photoionization. The data analysis was supported by energy calculations. RESULTS: Although the fragmentation patterns were basically the same at 270 eV and 330 eV, it was observed that above the C 1s edge the contribution to the spectrum from lower mass/charge fragment ions increased, pointing to a higher degree of dissociation of the molecule. Projections of the PEPIPICO spectra demonstrated the existence of unstable doubly charged species. The Gibbs free energy was calculated using the Møller-Plesset perturbation theory (MP2) for the neutral, singly and doubly excited camphor molecule. CONCLUSIONS: Our PEPIPICO spectrum clearly demonstrated the formation of doubly ionic dissociative species. From a slope analysis, we propose a secondary decay after a deferred charge separation mechanism in which, after a few steps, the camphor dication dissociates into C2 H3 (+) and C3 H5 (+) . This is the main relaxation route observed at 270 eV and 330 eV. The large energy difference between the mono and the dication (of the order of 258.2 kcal/mol) may explain the experimentally observed absence of stable dications in the spectra, because their formation is disadvantaged energetically.

Positive and negative ion formation in deep-core excited molecules: S 1s excitation in dimethyl sulfoxide.

The photo-fragmentation of the dimethyl sulfoxide (DMSO) molecule was studied using synchrotron radiation and a magnetic mass spectrometer. The total cationic yield spectrum was recorded in the photon energy region around the sulfur K edge. The sulfur composition of the highest occupied molecular orbital's and lowest unoccupied molecular orbital's in the DMSO molecule has been obtained using both ab initio and density functional theory methods. Partial cation and anion-yield measurements were obtained in the same energy range. An intense resonance is observed at 2475.4 eV. Sulfur atomic ions present a richer structure around this resonant feature, as compared to other fragment ions. The yield curves are similar for most of the other ionic species, which we interpret as due to cascade Auger processes leading to multiply charged species which then undergo Coulomb explosion. The anions S(-), C(-), and O(-) are observed for the first time in deep-core-level excitation of DMSO.

Absolute electron impact ionization cross-sections for CF4: Three dimensional recoil-ion imaging combined with the relative flow technique.

Here we present measurements of dissociative and non-dissociative cross-sections for the electron impact of the CF4 molecule. The present experiments are based on a Recoil Ion Momentum Spectrometer (RIMS), a standard gas mixing setup for CF4, and a reference gas. The measurements were carried out at several electron energies up to 1 keV, covering the energy range of previous experiments. We apply the relative flow technique (RFT) to convert the relative cross-sections measured by the RIMS into absolute values. Using the combination of RIMS and RFT, ion collection and calibration errors were minimized. The results were compared with theoretical and experimental studies available in the literature. Previous electron impact experiments present relative cross-sections or use correction terms for the absolute cross-sections due to losses of energetic ions. We elucidate the differences between the new measurement method and the existing ones in the literature and explain why the present method can be considered reliable. Furthermore, we show how reducing correction terms affects the results.

Core level (S 2p) excitation and fragmentation of the dimethyl sulfide and dimethyldisulfide molecules.

Electronic excitation and ionic dissociation of dimethylsulfide (DMS) and dimethyldisulfide (DMDS) have been studied around the S 2p edge using synchrotron radiation and time-of-flight mass spectrometry techniques. Mass spectra were obtained for both molecules, below, on and above the well defined resonances observed in the S 2p photoabsorption spectrum and centered at approximately 166 eV photon energy. Ab initio IS-CASSCF calculations were performed for a better understanding of the photoabsorption spectra. Similar calculations were also performed for the H(2)S molecule, in order to establish a bench mark. For both molecules, a higher fragmentation degree is observed with increasing photon energy. In the DMDS case, selective fragmentation was observed in the formation of the [CH(n)S](+) ions at the first S 2p resonance (corresponding to excitation to a σ*SS state) and in the formation of the [S(2)](+) and [S](+) ions at the third S 2p resonance (corresponding to excitation to a σ*CS state). Previously unreported doubly charged ([S](2+), [CH(3)](2+)) are observed for DMS and DMDS.

The use of bone marrow cells grown in long-term culture for autologous bone marrow transplantation in acute myeloid leukaemia: an update.

Eleven patients with acute myeloid leukaemia have been transplanted with autologous marrow grown in long-term bone marrow culture. In the high risk group (six patients who had all previously relapsed) the procedure induced a remission in two patients who were in florid relapse at the time of the transplant. Five patients were transplanted in first remission and they remain well and disease-free between 150 and 12 weeks after their autologous transplant.

Autografting in Philadelphia (Ph)+ chronic myeloid leukaemia using cultured marrow: an update of a pilot study.

Incubation of CML marrow in long-term culture (LTC) conditions may result in selection of normal (Ph-) LTC-initiating cells (LTC-IC) as early as 10 days, and in production of Ph- clonogenic cells and mature end cells within 5 weeks. This was the rationale for using marrow cells from 10-day-old LTC to autograft nine chronic phase CML patients, ineligible for HLA-matched sibling donor transplant, and who were selected on the basis of a pre-transplant screening LTC test. Of the transplanted patients three died; two of graft failure and one of therapy-related toxicity with 97% Ph- cells 16 months following the autograft. The reconstituting haemopoietic cells in the seven engrafted patients were 100% Ph- in four, > or = 90% Ph- in two and 71% Ph- in the seventh, with a duration of complete cytogenetic response of 6-12 months. Three patients reverted to chronic phase and 100% Ph+ haemopoiesis 27-36 months post-autograft. The other three patients remain in continuous haematological remission with 22% Ph- cells in one and complete cytogenetic remission in the other two 3-4 years post-autograft. IFN therapy was generally introduced on the first evidence of recurrence of Ph+ cells or of cytogenetic deterioration. Further strategies to modulate immune surveillance in vivo may improve the outcome of cultured marrow autografts which give an initial and rather prolonged bias towards Ph- haemopoiesis.

[The effect of GM-CSF/IL-3 on the grafting efficiency of CD34+ hemopoietic progenitor cells from umbilical cord blood].

CD34+ cells were isolated from human umbilical cord blood by using a high-gradient magnetic cell sorting system with anti-CD34 monoclonal antibody coated with microbeads, and then inoculated onto a pre-formed irradiated bone marrow stroma to evaluate the grafting efficiency of CD34+ cells with rhGM-CSF or IL-3, alone or in combination. The results showed that only 36% CD34+ cells in the control, and 68 - 89.6% in the groups with growth factors treatment seeded to the stroma. A short incubation of CD34+ cells with growth factors could rapidly reconstitute a long-term hemopoiesis in the long-term liquid culture system. This demonstrates that a brief treatment of CD34+ cells with GM-CSF or IL-3 can improve the grafting efficiency of transplanted umbilical cord blood cells.

Dissociative photoionization of adenine following valence excitation.

We present results on the valence level excitation, ionization and dissociation of adenine, using time-of-flight mass spectrometry and synchrotron radiation, in the vacuum ultraviolet (VUV) range of 12-21 eV. The measurements were performed using a gas-phase (Ne) harmonics filter in order to eliminate contributions from higher-order harmonics. Mass spectra were obtained using the photoelectron-photoion coincidence technique (PEPICO). The relative abundances for each ionic fragment and their mean kinetic energy release have been determined from the analysis of the corresponding peak shapes in the mass spectra. Comparison with the available photoelectron spectra and previous measurements allowed the assignment of the main features in the spectra. A discussion on the dissociative photoionization channels of this molecule has also been included. Due to our harmonics-free incident photon beam we were able to propose new appearance energy (AE) for the most important ionic channels in this energy range. The precursor ion, C(5)H(5)N(5)+, is the most abundant species (40% at 15 eV and 20% at 20 eV), which confirms the high stability of adenine upon absorption of VUV photons. We have observed other intense fragment ions such as: C(4)H(4)N(4)+, C(3)H(3)N(3) (+), C(2)H(2)N(2)+ and HCNH+. The production of the neutral HCN fragment represents up to 40% of the dissociative channels for this molecule as induced by VUV photons.

The myelosuppressive effect of recombinant interferon gamma in short-term and long-term marrow cultures.

The effect of a highly purified human gamma IFN (r-gamma-IFN) on the growth of haemopoietic progenitors was examined in soft agar assays and in long-term bone marrow cultures. r-gamma-IFN reduced colony formation by progenitor cells of the granulocyte/macrophage lineage (GM-CFC), the erythroid lineage (BFU-E) and multipotent cells (GEMM-CFC), and suppressed haemopoiesis in long-term culture in a dose-related fashion. At high doses (1000 units/ml) r-gamma-IFN appeared to be toxic to the stromal cells of the bone marrow.

Lack of response of bone marrow, in vitro, to growth factors in congenital neutropenia.

Severe congenital neutropenia has a poor outlook. In vitro clonogenic assays using recombinant growth factors may improve understanding of the underlying pathogenetic mechanisms and identify those in whom growth factors might be clinically useful. Marrow from a boy with congenital neutropenia was cultured with a variety of recombinant growth factors. The results show that the neutropenia did not result from a lack of myeloid progenitors but that these progenitors could not produce mature neutrophils. Bone marrow transplantation is being considered as the most likely approach to correct neutropenia.

Functional studies of bone marrow haemopoietic and stromal cells in the myelodysplastic syndrome (MDS).

Long-term bone marrow culture (LTBMC) was used to investigate the proliferative behaviour of marrow cells from a spectrum of cases of the myelodysplastic syndrome (MDS), and the results compared with those obtained in the conventional short-term clonal assay. Two broad patterns of growth were revealed in LTBMC. In one group the incidence of haemopoietic progenitor cells steadily declined to abnormally low levels at 4 weeks, while in a second group they were maintained near normal levels for periods of up to 7 weeks. These growth patterns, which were not predictable from clonogenic assays on the marrow cells prior to LTBMC, or from the morphology of the bone marrow, may reflect the stage of evolution of the disease. Further studies of clonality are required to establish whether or not patients exhibiting the second pattern have a potentiality to harbour residual normal haemopoiesis. LTBMC was also used to study the function of MDS marrow stroma in terms of its ability to sustain the growth of normal haemopoietic progenitor cells. Although the phenotype of the cultured adherent cell layer, obtained from some patients, was atypical, no consistent functional defect of MDS stroma could be identified by studying the level of haemopoiesis reached by normal cells seeded into MDS stroma.

The effect of the GM-CSF/IL-3 fusion protein PIXY321 on bone marrow and circulating haemopoietic cells of previously untreated patients with cancer.

This is a phase I/II study of the GM-CSF/IL-3 fusion protein (PIXY321. Patients were treated with PIXY321 at a daily subcutaneous dose of 500, 750 and 1000 micrograms/m2 for 14 d. Side-effects were mild and consisted mainly of injection-site reactions and constitutional symptoms. A biphasic modest increase of white blood count (2-5-fold) and platelets (1-1.5 fold) was seen, accompanied by an increased bone marrow cellularity and an increase in circulating progenitors. Colony-forming cells in the blood rose to a median of 184 granulocyte/macrophage-colony forming cells (GM-CFC)/ml, eight Mix-CFC/ml, 250 burst forming units-erythroid (BFU-E)/ml and 140 CFU-mega-karyocytes/ml, corresponding to a 10-, 2.5, 8- and 30-fold increase respectively. When seeded for long-term culture on irradiated bone marrow stroma, the mobilized cells were not able to sustain haemopoiesis in vitro to the same degree as bone marrow. Taken together these results indicate that PIXY321 has a biological effect in humans more similar to that of IL-3 than to that of GM-CSF.

Extensive amplification of single cells from CD34+ subpopulations in umbilical cord blood and identification of long-term culture-initiating cells present in two subsets.

CD34+ cord blood cells were isolated with immunomagnetic beads and fractionated by fluorescence-activated cell sorting (FACS) into three subpopulations: CD34+38+DR+, CD34+38-DR+ and CD34+38-DR-, using antibodies specific for these cell surface markers. Cells from each of the three subsets were plated as single cells in serum-free medium supplemented with a combination of growth factor and individual cells were monitored for proliferation and the capacity to form colony-forming cells. Single cells from the CD34+38+DR+ subset showed the lowest expansion capacity, generating up to 1.1 x 10(6) cells at five weeks, while individual cells from both the CD34+38-DR+ and CD34+38-DR- subsets could be expanded up to 1.8 x 10(6) and 9.2 x 10(6) cells, respectively, over a period of six weeks. The different subpopulations also generated colony-forming cells which gave rise to erythroid, myeloid and erythroid/myeloid colonies. CD34+38-DR+ cells generated large numbers of colonies within two weeks in liquid culture, but this rapidly declined. Generation of lineage-committed colony-forming cells was better sustained in the CD34+38-DR- population and continued for up to six weeks in culture. Overall, the generation of colony-forming cells declined with time in culture, although the cell numbers continued to expand. However, when the same populations were plated on irradiated bone marrow stroma, both the CD34+38-DR+ and the CD34+38-DR- cells were capable of producing granulocytemacrophage colony-forming cells (GM-CFCs) for 10 to 12 weeks. As hemopoiesis was sustained for almost three months, it appears that these populations were significantly enriched in long-term culture-initiating cells (LTC-ICs). Although both populations generated GM-CFCs, the CD34+38-DR- cells sustained production of higher numbers of colony-forming cells than the CD34+38-DR+ population. These results demonstrate that cells from cord blood can be efficiently monitored at the single-cell level for proliferation, expansion and colony-forming capacity. Furthermore, at least two populations of LTC-ICs can be distinguished in cord blood CD34+38- cells by the differential expression of the HLA-DR antigen.

Effects of recombinant human granulocyte colony-stimulating factor (CSF), human granulocyte macrophage-CSF, and gibbon interleukin-3 on hematopoiesis in human long-term bone marrow culture.

We have studied the effects of recombinant human granulocyte colony-stimulating factor (rhG-CSF), hG macrophage-CSF (hGM-CSF), and gibbon interleukin-3 (gIL-3) on cell proliferation and differentiation in human long-term bone marrow culture (LTBMC). hG-CSF induced a maximal increase of 2.3-fold in both total nonadherent cells and GM cluster-forming cells, but only an increase of 1.7-fold in GM-colony-forming cell (GM-CFC) numbers, influencing mainly neutrophil differentiation. Cultures treated with hGM-CSF demonstrated a peak of 12.8-, 21- and 3.2-fold elevations in total nonadherent cells, cluster, and GM-CFC, respectively, and influenced differentiation of neutrophils, monocytes, eosinophils, and lymphocytes. Cultures treated with gIL-3 demonstrated the largest expansion in the GM-CFC population, reaching a maximum of 5.3-fold in relation to that of unstimulated controls. IL-3 treatment also increased the numbers of GM clusters and mature cells (including all myeloid cells and lymphocytes) 7.8- and 4.8-fold, respectively. Similar quantitative and qualitative changes were induced by G-CSF, GM-CSF, and IL-3 in LTBMCs of patients in remission after treatment for acute lymphoblastic leukemia or Hodgkin's lymphoma. Overall, the expansion of GM progenitor cells in cultures treated with growth factors was larger in the adherent cell layer than in the nonadherent cell fraction. In addition, hGM-CSF, gIL-3, and hG-CSF to a less extent, increased the cycling rates of GM-CFC progenitors located in the adherent layer. These results indicate that hG-CSF is a much less potent stimulus of hematopoiesis in LTBMC than the other CSFs assayed, and that the increases in cell production after treatment with G-CSF, GM-CSF, or IL-3 may be achieved by primary expansion of different cell populations within the hierarchy of the hematopoietic system. The effects of the growth factors were transient and the longevity of hematopoiesis in the cultures was not altered, suggesting that treatment with IL-3, GM-CSF, or G-CSF had not compromised the ability of primitive cells to give rise to mature cells. This indicates that the stromal microenvironment in LTBMC can override potential differentiation-inducing activities of the CSFs.

The use of cultured bone marrow cells in autologous transplantation.

The feasibility of ex vivo purging with long-term bone marrow cultures (LTBMC) for autologous transplantation in leukemia has been established. The procedure has been applied to patients with acute myeloid leukemia (in relapse or remission) and recently in one patient with chronic myeloid leukemia. The results in first remission AML are very encouraging with 4 out of 6 patients well and apparently disease free greater than 1 to 4 years post autograft. In patients transplanted with active disease, remissions of 6 to 8 months duration were seen in two patients transplanted in florid relapse. In a CML patient with 87% of his bone marrow cells Ph1 positive, there was a marked decline of karyotypically abnormal cells in culture. Reinfusion of the cultured cells into the patient resulted in engraftment with exclusively Ph1 negative cells. The therapeutic implications of LTBMC purging require further evaluation.

Growth of normal versus leukemic bone marrow cells in long term culture from acute lymphoblastic and myeloblastic leukemias.

The ability of the in vitro long-term bone marrow culture (LTBMC) system to impair the survival of leukemic cells and to enhance the growth of normal progenitors has been studied. Bone marrow cells from 19 acute lymphoblastic leukemia (ALL) and 30 acute myeloid leukemia (AML) patients at diagnosis were grown in LTBMC for 4-10 weeks. In half of the cases the leukemic population declined down to undetectable levels and was replaced by putative normal hemopoietic precursors, both in ALL and in AML. In the remaining cases, leukemic cells persisted throughout the culture time and few if any normal hemopoietic cells were detected. These data led us to extend to the lymphoid compartment the previous observation of decreasing leukemic myeloid blasts in LTBMC. The potential of such cultures as an in vitro purging system for autologous bone marrow transplantation in selected poor-prognosis lymphoid malignancies should be explored, as has been done for acute and chronic myeloid leukemias.

Residual Ph- cells in chronic myeloid leukemia: detection and usefulness.

Bone marrow cells from patients with chronic myeloid leukemia (CML) in in vitro long-term bone marrow culture (LTBMC) show an impaired survival of Philadelphia (Ph) positive cells and, in a proportion of patients, the emergence of Philadelphia negative hemopoiesis. The standard conditions of in vitro cultures provide optimal purging effect. Selected patients can now have autologous bone marrow transplants (ABMT) with in vitro purged cells.